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Abstract

Glutamate is a neurotransmitter critical for spinal excitatory synaptic transmission
and for generation and maintenance of spinal states of pain hypersensitivity via activation
of glutamate receptors. Understanding the regulation of synaptically and non-synaptically
released glutamate associated with pathological pain is important in exploring novel
molecular mechanisms and developing therapeutic strategies of pathological pain. The
glutamate transporter system is the primary mechanism for the inactivation of synaptically
released glutamate and the maintenance of glutamate homeostasis. Recent studies demonstrated
that spinal glutamate transporter inhibition relieved pathological pain, suggesting
that the spinal glutamate transporter might serve as a therapeutic target for treatment
of pathological pain. However, the exact function of glutamate transporter in pathological
pain is not completely understood. This report will review the evidence for the role
of the spinal glutamate transporter during normal sensory transmission and pathological
pain conditions and discuss potential mechanisms by which spinal glutamate transporter
is involved in pathological pain.

Review

In addition to its essential metabolic role, glutamate is a major mediator of excitatory
signals in the central nervous system and is involved in many physiologic and pathologic
processes, such as excitatory synaptic transmission, synaptic plasticity, cell death,
stroke, and chronic pain [1,2]. Glutamate exerts its signaling role by acting on glutamate receptors, including
N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate,
and metabotropic glutamate receptors. These receptors are located on the pre- and
post-synaptic membranes, as well as, at extra-synaptic sites. Glutamate concentration
in the synaptic cleft determines the extents of receptor stimulation and excitatory
synaptic transmission. It is of critical importance that the extracellular glutamate
concentration be kept at physiological levels, as excessive activation of glutamate
receptors can lead to excitotoxicity and neuronal death [3]. The clearance of glutamate from the synaptic cleft is principally dependent on Na+-dependent, high-affinity, neuronal glutamate transporters present presynaptically,
postsynaptically, and perisynaptically, and on glial glutamate transporters (Fig.
1). Currently, five isoforms of glutamate transporters have been identified [3]: namely, GLAST (glutamate/aspartate transporter), GLT-1 (glutamate transporter-1),
EAAC (excitatory amino acid carrier) 1, EAAT (excitatory amino-acid transporter) 4,
and EAAT5. The human homologues of the three more ubiquitous subtypes (GLAST, GLT-1,
and EAAC1) are named EAAT1, EAAT2, and EAAT3, respectively. The five isoforms belong
to the same gene-family and share 50–60% amino acid sequence identity [3]. However, they have discrete cellular and regional localizations. GLAST is present
in glial cells throughout the central nervous system, with strong labeling in cerebellar
Bergmann glia and more diffuse labeling in the forebrain [3]. It is also transiently expressed in a small number of neurons [4]. GLT-1 is almost exclusively expressed on glia and is widespread and abundant throughout
the forebrain, cerebellum, and spinal cord [4]. In contrast, EAAC1 is found predominantly in neurons of the spinal cord and brain
[4,5]. EAAT4 has properties of a ligand-gated Cl-channel and is localized mainly in cerebellar
Purkinje cells [6]. EAAT5 is retina-specific [7].

Figure 1. Glutamate (Glu) uptake and Glu/glutamine (Gln) cycle. Glu released from the nerve
terminal by exocytosis is taken up by neuronal Glu transporter present presynaptically
(1) and postsynaptically (2) and by glial Glu transporter (3). Glu/Gln cycle is one
type of Glu recycling, but the significance is still unclear in vivo (see references 37 and 38). Astroglia detoxifies Glu by converting it to Gln. Glu
is subsequently released from the glial cells by glial Gln transporter (4) and taken
up by neuronal Gln transporter (5). Neurons convert Gln back to Glu, which is loaded
into synaptic vesicles by vesicular Glu transporter (6). 7: postsynaptic Glu receptors.

Given the well-documented evidence that glutamate acts as a major excitatory neurotransmitter
in primary afferent terminals [2], it is expected that glutamate transporter might be involved in excitatory sensory
transmission and pathological pain. Indeed, recent studies have revealed that inhibition
of spinal glutamate transporter produced pro-nociceptive effects under normal conditions
[8] and have unexpected antinociceptive effects under pathological pain conditions [9-11]. It is not completely understood why the effects of spinal glutamate transporter
inhibition under pathological pain conditions are opposite to its effects under normal
conditions. In this review, we will illustrate the expression and distribution of
the glutamate transporter in two major pain-related regions: spinal cord and dorsal
root ganglion (DRG). We will also review the evidence for the role of the glutamate
transporter during normal sensory transmission and pathological pain conditions and
discuss potential mechanisms by which glutamate transporter is involved in pathological
pain.

Expression and distribution of glutamate transporter in the spinal cord and dorsal
root ganglion

In the spinal cord, three isoforms of glutamate transporter (GLAST, GLT-1, and EAAC1)
have been reported [4,12]. They are expressed in highest density within the superficial dorsal horn of the
spinal cords of rats and mice (Fig. 2). GLT-1 and GLAST are exclusively distributed in glial cells at perisynaptic sites
in the superficial dorsal horn [13]. EAAC1, in addition to its expression in the spinal cord neurons, is detected in
the DRG and distributed predominantly in the small DRG neurons (but not in DRG glial
cells) [12] (Fig. 3). Some of these EAAC1-positive DRG neurons are positive for calcitonin gene-related
peptide (CGRP) or are labeled by IB4 [12,13]. Unilateral dorsal root rhizotomy shows less intense EAAC1 immunoreactivity in the
superficial dorsal horn on the ipsilateral side, compared to the contralateral side
[12]. Moreover, confocal microscopy demonstrates that some EAAC1-positive, small dot-
or patch-like structures in the superficial laminae are labeled by IB4 or are positive
for CGRP [12]. Under electron microscope, EAAC1 is associated with the axon terminal and dendritic
membranes at synaptic and non-synaptic sites and is present with CGRP in the axons
and the terminals in the superficial dorsal horn [13]. The expression level and distribution pattern of neuronal and glial glutamate transporters
in the superficial dorsal horn suggest an important role for spinal glutamate transporter
in spinal nociceptive transmission. In addition, the unique expression of EAAC1 in
the small DRG neurons and nociceptive primary afferent terminals suggests that EAAC1
might have a distinct role in pain processing, compared to GLT-1 and GLAST.

Figure 2. Expression and distribution of the glutamate transporter in the dorsal root ganglion
(A) and the spinal cord (B-D). EAAC1 is expressed mainly in small dorsal root ganglion
cells (A) and distributed predominantly in the superficial dorsal horn of the spinal
cord (B). GLAST (C) and GLT-1 (D) are expressed highly in the superficial dorsal horn
and the region around the central canal. Scale bars: 10 μm in A and 125 μm in B, C,
and D.

Role of the spinal cord glutamate transporter in normal sensory transmission

Recently evidence suggests that spinal glutamate transporter might play an important
role in normal sensory transmission. Liaw et al. [8] reported that intrathecal injection of glutamate transporter blockers DL-threo-β-benzyloxyaspartate
(TBOA) and dihydrokainate (DHK) produced significant and dose-dependent spontaneous
nociceptive behaviors, such as licking, shaking, and caudally directed biting, phenomena
similar to the behaviors caused by intrathecal glutamate receptor agonists, such as
glutamate, NMDA, or AMPA, when given intrathecally [14-16]. Intrathecal TBOA also led to remarkable hypersensitivity in response to thermal
and mechanical stimuli [8]. These findings are consistent with a previous report that showed an increase in
spontaneous activity and responses of wide dynamic range neurons to both innocuous
mechanical (brush, pressure) and noxious mechanical (pinch) stimuli after topical
application of L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC), a glutamate transporter
blocker [17,18]. TBOA-induced behavioral responses could be significantly blocked by intrathecal
injection of the NMDA receptor antagonists MK-801 and AP-5, the non-NMDA receptor
antagonist CNQX or the nitric oxide synthase inhibitor L-NAME [8]. The effects of DHK and PDC were thought to be partially due to their non-specific
interactions with glutamate receptors. However, unlike DHK and PDC, TBOA does not
act as an agonist or antagonist at glutamate receptors [9,19,20]. Thus, spontaneous pain-related behaviors and sensory hypersensitivity evoked by
TBOA directly support the involvement of glutamate transporter in normal excitatory
synaptic transmission in the spinal cord. In vivo microdialysis analysis showed that intrathecal injection of TBOA produced short-term
elevation of extracellular glutamate concentration in the spinal cord [8]. Topical application of TBOA on the dorsal surface of the spinal cord also resulted
in a significant elevation of extracellular glutamate concentrations demonstrated
by in vivo glutamate voltametry [8]. These findings indicate that a decrease of spinal glutamate uptake can lead to excessive
glutamate accumulation in the spinal cord, which might, in turn, result in over-activation
of glutamate receptors, and production of spontaneous nociceptive behaviors and sensory
hypersensitivity. Thus, glutamate uptake through spinal glutamate transporters is
critical for maintaining normal sensory transmission under physiological conditions.

Expression and function of the spinal cord glutamate transporter in pathological pain
states

Glutamate uptake and expression of glutamate transporters in the spinal cord have
been found to be changed under pathological conditions associated with chronic pain
status. Chronic constriction nerve injury upregulated glutamate transporter expression
at day 1 and 4 postoperatively, but it downregulated glutamate transporter expression
at days 7 and 14 postoperatively [21]. Moreover, chronic constriction nerve injury significantly reduced spinal glutamate
uptake activity at day 5 postoperatively [21]. Recently, another study showed that spinal nerve ligation also markedly reduced
glutamate uptake activity, as demonstrated in spinal deep dorsal and ventral horn
4–6 weeks after the nerve ligation [22]. Although the underlying mechanism by which neuropathic inputs cause the decrease
in spinal glutamate uptake is unclear, it is thought that this decrease might contribute
to the central mechanisms of the development and maintenance of pathological pain[21,22].

As shown above, inhibition of glutamate uptake produces pronociceptive effects in
normal animals [8]. Unexpectedly, in pathological pain states, inhibition of glutamate transporter activity
produced antinociceptive effects. For example, glutamate transporter inhibitors attenuated
the induction of allodynia induced by PGE2, PGF2α, and NMDA [9]. Inhibition or transient knockdown of spinal GLT-1 led to a significant reduction
of nociceptive behavior in the formalin model [10]. Consistent with these findings, the preliminary work from Yuan-Xiang Tao's laboratory
showed that three different glutamate transporter inhibitors (TBOA, DHK, threo-3-hydroxyaspartate)
reduced formalin-induced nociceptive responses and Complete Freund's adjuvant (CFA)-evoked
thermal hyperalgesia [11]. On the other hand, the glutamate transporter activator MS-153, which is reported
to accelerate glutamate uptake in in vivo and in vitro studies [23-26], had no effect in formalin tests when MS-153 was applied via intrathecal injection,
even at the highest dose (1,000 μg/10 μl) [11]. Interestingly, Sung et al. reported that riluzole, a glutamate transporter regulator,
significantly attenuated thermal hyperalgesia and mechanical allodynia after chronic
constriction nerve injury [21], but this drug was ineffective against peripheral neuropathic pain in a clinical
setting [27]. The reason for the discrepancy between the two studies is unclear, but it is worth
noting that, in addition to increasing glutamate uptake, riluzole has multiple actions
on many systems [neuroprotective, anticonvulsant, anxiolytic, and anesthetic qualities
by its blockade of sodium channel α-subunits, glutamate receptors, and γ-aminobutyric
acid (GABA) reuptake and its stabilization of voltage-gated ion channels] [28-31]. Thus, more selective drugs that promote spinal glutamate transporter function are
needed to demonstrate whether glutamate transporter activators have possible efficacy
in the treatment of chronic pain.

The intriguing question remains as to why glutamate transporter inhibitors have antinociceptive
effects under pathologic pain conditions that are opposite to their pro-nociceptive
effects under normal conditions. Several mechanisms potentially contribute to the
role of the glutamate transporter inhibitors under pathological pain states (Fig.
4). First, the blockade of spinal glutamate transporter uptake inhibits clearance of
glutamate, leading to the chronic elevation of spinal extracellular glutamate, possibly
subsequently causing excitotoxicity, compromising or destroying subsceptible dorsal
horn neurons, and interfering with the transmission of pain signaling. However, preliminary
data from Dr. Tao's laboratory showed that transient glutamate transporter inhibition
did not produce significant spinal neuronal damage in rat formalin or CFA model [11]. Second, GABA, an inhibitory transmitter, is synthesized from glutamate by glutamic
acid decarboxylase. Do the increased glutamate levels caused by glutamate transporter
inhibition lead to increased GABA in the spinal cord? Recent work showed that inhibition
of glutamate transporter activity depleted both glutamate and GABA neurotransmitter
pools and reduced inhibitory postsynaptic current (IPSC) and miniature IPSC amplitudes
[32,33]. Thus, if this property extends to the spinal cord, one would expect that blockade
of spinal glutamate transporter would decrease the amount of GABA in GABAergic terminals
and reduce IPSP or IPSC. This expectation would not explain the mechanisms of antinociception
by glutamate transporter blocker in pathological pain. Third, inhibition of reuptake
through presynaptic EAAC1 and/or the glutamate/glutamine cycle in the spinal cord
might result in a depletion of glutamate in synaptic vesicles and a decrease in presynaptically
released glutamate, leading to a reduction in glutamate receptor-mediated nociceptive
transmission. It is documented that abundant glutamate is distributed in intracellular
space, particularly inside nerve terminals [3,34,35]. As a precursor for transmitter glutamate, glutamine is also rich in the intracellular
and extracellular fluid [3]. Moreover, although it has been demonstrated in vitro that glutamine is a precursor of transmitter glutamate, the in vivo evidence regarding the glutamate/glutamine cycle is less convincing [36]. The ability of glutamatergic neurons to sustain release of glutamate independently
of glutamine might be related to a newly found capacity for pyruvate carboxylation
[37,38]. Pyruvate carboxylation replenishes the loss of α-ketoglutarare from the tricarboxylic
acid cycle that is inherent in release of glutamate. Thus, inhibition of spinal glutamate
transporter might not cause the depletion of glutamate in presynaptic vesicles in
pathological pain. Fourth, chronic elevation of extracellular glutamate caused by
glutamate uptake inhibition might activate the inhibitory presynaptic metabotropic
glutamate receptors (mGluRs) [39-41] and promote a postsynaptic desensitization of glutamate receptors [40]. It is possible that, during pathological pain conditions, glutamate transporter
inhibitor-produced antinociception might be due to the decreased release of pre-synaptic
glutamate via activation of inhibitory mGluRs in primary afferent terminals and/or
reduced postsynaptic efficacy of glutamate via desensitization of glutamate receptors
in the spinal dorsal horn neurons. Finally, glutamate transporter inhibitors might
produce antinociception in pathological pain by the mechanism of blocking inverse
operation of the glutamate transporter. It is well documented that the glutamate transporter
imports one glutamate ion and co-transports three Na+ ions into the cell [42] and that transporter function is dependent upon both the membrane potential and the
transmembrane ion gradients established by Na+-K+ATPase as driving forces [43,44]. Under physiological conditions, these forces are sufficient to maintain the concentration
gradient of micromolar extracellular glutamate against millimolar intracellular glutamate
through glutamate transporter uptake [3,42]. However, under pathological conditions, metabolic insults that deplete intracellular
energy and alter ionic gradients can lead to reversed action of the glutamate transporter
[3]. For example, during brain ischemia, ATP is depleted and impairment of Na+-K+ATPase results in the increases in intracellular Na+ ions and extracellular K+ ions, which causes inverse operation of the glutamate transporter and release of glutamate
into the extracellular space [3]. Indeed, the glutamate transporter inhibitors (e.g., TBOA) reduce glutamate release
and have neuroprotective actions in brain ischemia [20]. Does pathological persistent pain cause cellular energy insufficiency that inverses
the glutamate transporter operation to release glutamate in the spinal cord? Metabolic
activity and energy demand significantly increase in the spinal cord under pathological
pain conditions [45-48]. Such hyperactive states of spinal neuronal and glial cell activities might not only
consume large amounts of cell energy, but also disturb energy metabolism, decrease
ATP, and result in energy insufficiency that might reverse spinal glutamate transporter
operation to release glutamate. It is possible that blocking the reversed glutamate
transporter-mediated glutamate release is an underlying mechanism of antinociception
produced by glutamate transporter inhibition under chronic pain conditions.

Taken together, it is evident that at least five potential mechanisms are involved
in the action of glutamate transporter inhibitors during pathological pain (Fig. 4). In the first four mechanisms, glutamate transporter inhibitors lead to an increase
in spinal extracellular glutamate levels, whereas, in the last one, glutamate transporter
inhibitors block the reversed glutamate transporter-mediated glutamate release, and
reduce extracellular glutamate levels (Fig. 4). Therefore, two distinct models explain the role of spinal glutamate transporter
in pathological pain (Fig. 4). Determining extracellular glutamate levels in the spinal cord following glutamate
transporter inhibition during pathological pain might be a key to determine the mechanisms
of glutamate transporter inhibitor-produced antinociception in the state of pathological
pain.

Conclusion

Pathological pain, particularly as a result of nerve injury, is poorly managed by
current drugs, such as opioids and non-steroidal anti-inflammatory drugs. Glutamate
receptor antagonists are effective in reducing pain hypersensitivity in animal models
and clinical settings, but with unacceptable side effects. Glutamate transporter inhibitors
have recently been shown to produce antinociceptive effects in several preclinical
pathological pain models. Further studies to delineate the role of the spinal glutamate
transporters during chronic pain states might lead to better strategies for the prevention
and therapy of chronic pain.

Acknowledgements

This work was supported by the Johns Hopkins University Blaustein Pain Research Fund
and in part by NIH grant NS44219. The corresponding author would like to thank Drs.
John A. Ulatowski and Roger A. Johns for their support. The authors thank Tzipora
Sofare, MA, for her editorial assistance.